Although in recent years significant advances have been made in carbohydrate chemistry, there are still substantial difficulties associated with the chemical synthesis of glycoconjugates, particularly with the formation of the ubiquitous β-1,2-cis-mannoside linkage found in mammalian oligosaccharides. Moreover, regio- and stereo-chemical obstacles must be resolved at each step of the de novo synthesis of a carbohydrate.
In view of the difficulties associated with the chemical synthesis of glycoconjugates, the use of glycosyltransferases to enzymatically synthesize glycoproteins and glycolipids, having desired oligosaccharide moieties, is a promising approach to preparing such glycoconjugates. Enzyme-based syntheses have the advantages of regioselectivity and stereoselectivity, and can be performed using unprotected substrates. Moreover, glycosyltransferases have been used to enzymatically modify oligosaccharide moieties and have been shown to be very effective for producing specific products with good stereochemical and regiochemical control. The glycosyltransferases of interest include fucosyltransferases, sialyltransferases, galactosyltransferases, and N-acetylglucosaminyltransferases. For a general review, see, Crout et al., Curr. Opin. Chem. Biol. 2: 98-111 (1998) and Arsequell, et al., Tetrahedon: Assymetry 10: 2839 (1997).
Many glycoproteins and glycolipids require the presence of a particular glycoform, or the absence of a particular glycoform, in order to exhibit a particular biological activity. For example, many glycoprotein and glycolipids require the presence of particular fucosylated structures in order to exhibit biological activity. Intercellular recognition mechanisms often require a fucosylated oligosaccharide. For example, a number of glycoproteins that function as cell adhesion molecules, including P-selectin, L-selectin, and E-selectin, bind specific cell surface fucosylated carbohydrate structures such as the sialyl Lewis-x and the sialyl Lewis-a structures. In addition, the specific carbohydrate structures that form the ABO blood group system are fucosylated. The carbohydrate structures in each of the three groups share a Fucα1,2Galβ1-disaccharide unit. In blood group O structures, this disaccharide is the terminal structure; whereas the blood group A structure is formed by an α1,3 GalNAc transferase that adds a terminal GalNAc residue to the disaccharide; and the blood group B structure is formed by an α1,3 galactosyltransferase that adds a terminal galactose residue.
The Lewis blood group structures are also fucosylated. For example the Lewis-x and Lewis-a structures are Galα1,4(Fucα1,3)GlcNac and Galβ1,3(Fucα1,4)GlcNac, respectively. Both these structures can be further sialylated (NeuAcα2,3-) to form the corresponding sialylated structures. Other Lewis blood group structures of interest are the Lewis-y and Lewis-b structures which are Fucα1,2Galβ1,4(Fucα1,3)GlcNAcβ-OR and Fucα1,2Galβ1,3(Fucα1,4)GlcNAc-OR, respectively. For a description of the structures of the ABO and Lewis blood group structures and the enzymes involved in their synthesis see, Essentials of Glycobiology, Varki et al. eds., Chapter 16 (Cold Spring Harbor Press, Cold Spring Harbor, N.Y., 1999).
Specifically, fucosyltransferases have been used in synthetic pathways to transfer a fucose residue from guanosine-5′-diphosphofucose to a specific hydroxyl of a saccharide acceptor. A variety of donor substrates and acceptor substrates are known (see Guo et al., Applied Biochem. and Biotech. 68: 1-20 (1997)). For example, Ichikawa prepared sialyl Lewis-x by a method that involves the fucosylation of sialylated lactosamine with a cloned fucosyltransferase (Ichikawa et al., J. Am. Chem. Soc. 114: 9283-9298 (1992)). Lowe has described a method for expressing non-native fucosylation activity in cells, thereby producing fucosylated glycoproteins on cell surfaces, etc. (U.S. Pat. No. 5,955,347).
Thus, since the biological activity of many commercially important recombinantly and transgenically produced glycoproteins and glycolipids depends upon the presence of a particular glycoform, or the absence of a particular glycoform, a need exists for an efficient method for enzymatically synthesizing glycoconjugates having the desired fucoylated oligosaccharide moieties. In addition, there is a need for the efficient production of focosylated oligosaccharides. The present invention fulfills these and other needs.